CN102202692B - Method for diagnosing cancers expressing HER2 receptor or its truncated variants - Google Patents

Method for diagnosing cancers expressing HER2 receptor or its truncated variants Download PDF

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CN102202692B
CN102202692B CN200980126660.1A CN200980126660A CN102202692B CN 102202692 B CN102202692 B CN 102202692B CN 200980126660 A CN200980126660 A CN 200980126660A CN 102202692 B CN102202692 B CN 102202692B
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祖亚昆·阿瑞巴斯·洛佩兹
吉姆·佩德森
皮尔·戴维·安吉丽妮
约瑟夫·路易斯·帕拉·帕劳
梭尔·劳斯
约瑟·巴塞尔加·托雷斯
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FUNDACIO PRIVADA INST D INVEST
FUNDACIO PRIVADA INSTITUCIO CA
FUNDACIO PRIVADA INST DE RECER
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Abstract

The present invention relates to a method of diagnosis and therapy of cancers expressing the HER2 receptor. The invention provides antibodies or fragments thereof that recognises an epitope of a HER2 receptor truncated form, said epitope being defined by a sequence included in SEQ ID NO: 2. The invention also provides a method of cancer diagnosis, which comprises the detection of the presence of the HER2 receptor truncated form consisting of the amino acid sequence SEQ JD NO: 1 in a patient sample.

Description

For diagnosing the method for the cancer of expressing HER2 acceptor or its clipped form
The present invention relates to the method for diagnosis expression HER2 acceptor cancer from separated sample.The present invention is also provided for the compound of early diagnosis and evaluation and the treatment of cancer.
Background technology
HER2 (claiming again c-erbB2, ErbB2 or Neu) is I type transmembrane protein, belongs to EGF-R ELISA or EGFR family, also referred to as HER1 or ErbB1.This family also comprises other two member HER3 and HER4.When HER1, HER3 or HER4 and EGF-type ligand binding, its ectodomain (domain) presents the configuration of " open ", forms homodimer and heterodimer.Because its ectodomain is " open " configuration, though it not with any ligand binding, the HER acceptor that HER2 also can be connected with part with other interacts.
The dimeric formation being caused by ectodomain caused HER receptor kinase in born of the same parents to interact, subsequently some tyrosine residues generation transphosphorylations.These Tyrosine O-phosphates, as Tyrosine O-phosphate in one group of born of the same parents-in conjunction with protein coupled device (couplers).The interaction occurring in cytoplasmic membrane reaches in nucleus by different signal transduction pathways, the protein kinase approach for example being activated by mitogen activated protein kinase (MAPK), the protein kinase approach (JNK) being activated by stress, Phospholipase C-gamma etc.All these signal circuits are being controlled genetic expression, in the mode of coordinating, modify, and play a role to determine the state of cell, for example cell proliferation, migration, survival and adhesion.Therefore, according to cell background, the activation of HER acceptor causes obvious cell response, and making cell transformation is to the cell between early ageing cell between cancer cells.
Except classical signal transduction pattern, HER acceptor or its fragment can form cell content and be transported to nucleus, the expression that they can direct regulation and control specific gene in nucleus.Observed HER2 this special case (Wang etc., " Binding at and transactivation of the COX-2promoter by nuclear tyrosine kinase receptor ErbB-2 ", cancer Cell-2004vol.6, pp.251-26), and this situation of C end fragment (CTF), it is comprised of HER4 acceptor clipped form, comprise whole tenuigenin part (Linggi etc., " ErbB-4 s80 intracellular domain abrogates ETO2-dependent transcriptional repression " j.Biological chemistry-2006, Vol.281, pp.25373-25380).
In human breast carcinoma, found a series of C end fragments or CTFs, infer membrane-spanning domain and tenuigenin territory that it contains HER2.(Molina etc., " NH (2)-terminal truncatedHER2 protein but not full-length receptor is associated with nodal metastasis in human breast cancer ", clinical Cancer Research-2002, Vol.8, pp.347-353).Also known suffer from the patient of expression HER2CTFs mammary cancer or suffer from the patient who expresses identical clipped form (it does not contain the N-end of HER2 (HER2CTFs)) mammary cancer, possibility that the breast cancer patients of suffering from main expression HER2 complete form than those develops into cancer metastasis larger 2002-supra such as () Molina and the poorer (Saez etc. of prognosis, " p95HER2 predicts worse outcome in patients with HER2-positive breast cancer " clinical Cancer Research-2006, Vol.12, pp.424-431).
Therefore, it is very important detecting in time the existence of HER2 in tumour, the more important thing is and detects it for complete form or brachymemma (CTF) form.
Now, in the aspect of routine clinical test, use the existence of antibody test HER2 complete form, to determine the type of problematic breast tumor.If detect that HER2 deposits, the therapy of recommendation is the trastuzumab (trastuzumab) of administering therapeutic monoclonal antibody ，Ru Genentech company.It is all that the purposes that this monoclonal antibody is used for the treatment of cancer is described in WO8906692 Zhong， Genentech company.In WO8906692, having described mono-clonal or polyclonal antibody directive action and matched in ，Gai extracellular region, the extracellular region of HER2 and the complete external region of albumen, is its N-end.As previously described, the epitope by antibody recognition of mentioning in WO8906692 is the antigen position of the ectodomain of HER2 complete form, does not comprise the C end fragment of clipped form or HER2.Therefore the antibody of, describing in WO8906692 and diagnostic method can not detect the existence of the HER2 of brachymemma.In addition, in the breast tumor of expressing at the CTFs of described HER2, antibody as trastuzumab be not curative because any epitope of their nonrecognition.This facts explain with the patient that HER2 clipped form (CTFs) is expressed in trastuzumab treatment, there is resistance.
The poor prognosis quantity of observing for preventing 2006 (supra) such as Saez, should adopt selectivity therapy for treating to express the patient of HER2 clipped form or CTFs.The patient who expresses HER2 clipped form tumour for detecting as early as possible (diagnosis) and treatment, the expression-form of distinguishing HER2 is very significant.So that successive treatment.
Anido etc. " Biosynthesis of tumorigenic HER2C-terminal fragments by alternative initiation of translation ", european Molecular Biology organization (EMBO) Journal-2006vol.25, pp.3234-3244, in document, described except acted on the fragment of HER2 generation by alpha-secretase enzyme, it produces the P95 of clipped form (CTF), the transmembrane segment that P95 contains acceptor and tenuigenin fragment, from being positioned at the mechanism of two initial translations of methionine(Met) of upstream and downstream, also produce respectively the clipped form (CTF) of two kinds of HER2 cross-film functional domains by optionally.Particularly, the aminoacid sequence that in the UniGene database of the U.S. state-run biotechnology information center (NCBI), accession number is M11730.1, is that optionally the initial translation of the methionine(Met) from 611 and 687 obtains.The optional form of also pointing out these HER2 acceptors (CTFs) in the document is present in breast tumor.Especially, it points out that a large amount of forms that exist are CTF687, or in other words, the albumen that the initial translation of methionine(Met) from 687 obtains.Anido etc., propose the tyrosine kinase activity inhibitor of HER2, and for example lapatinibditosylate (lapatinib) is used for the treatment of so that express the growth of these HER2 clipped form tumours and be down to minimum.Tyrosine kinase inhibitor is interacted and is brought into play effect by the C-end with HER2 acceptor, and described HER2 acceptor is to exist with the complete form of acceptor with by its CTFs form derivative or that produce by the initial translation of selectivity.
The document of Scaltriti etc. " Expression of p95HER2, a truncated form of the HER2 Receptor, and response to anti-HER2 therapies in breast cancer ", journal of National Cancer Institute-2007, Vol.99, pp.628-368 " in described for detection of the process for selective of a kind of existence in HER2 acceptor clipped form and learned case study, and (Trastuzumab, Herceptin) treatment produces the possible cause of resistance with trastuzumabs to list some.Especially, it emphasizes not have the p95HER2 fragment (product of HER2 after the hydrolysis of alpha-secretase zymoprotein) of the ectodomain of trastuzumab identification and the deposition of other this receptor clipped form.Scaltriti has proposed the immunofluorescence technique of a kind of detection p95HER2 fragment (product after the hydrolysis of alpha-secretase zymoprotein).This new detection method can be carried out on the tissue slice in being embedded in paraffin, and fixes with formalin according to clinical manipulation.This new methodology is derived from and observes p95HER2 clipped form, but not the complete form of acceptor, is arranged in cytoplasmic membrane and the tenuigenin of cell.More this method proposes by the anti-HER2 antibody of the tenuigenin functional domain with bind receptor, tenuigenin to be measured to be dyeed, have p95HER2 to express; And with anti-these results of cytokeratin antibody test result verification, the widely distributed instrument that is used as diagnosing tumour of this albumen.Yet, it be unclear that whether this method can express those tumour of HER2 complete form effectively and the tumor area of expression clipped form separates.
Be necessary to find new and effectively for the diagnosis and treatment of target, accurately relevant to the type of problem cancer, to can take in time effective methods for the treatment of to treat the poorest tumour of those prognosis, abandon those and do not have from the beginning resultful therapy.
The invention provides solution to the problems described above and to cancer, particularly mammary cancer is carried out the novel method of early stage classification.
Summary of the invention
Determine, a kind of existence of the HER2 clipped form (CTF) being produced by the initial translation of selectivity, very relevant to the prediction of the cancer types for the treatment of, this prescribes doctor and becomes easy in therapeutic process.The determined sequence with SEQID NO:1 of this HER2 clipped form (CTF-611).
This sequence has been used to develop multiple types of tools, to detect existing of HER2 clipped form in tissue samples, thereby provides a kind of new, diagnostic device reliably, also can be used for clinical diagnosis.
The present invention also provides new antibodies or its fragment of identification HER2 clipped form or CTF (it can not be identified by trastuzumab).The epitope that the sequence that these antibody recognition are comprised by SEQ ID NO:2 limits.The present invention also provides the hybridoma cell line that is suitable for producing this antibody.Obtainable anti-HER2 antibody recognition CTFs and HER2 or only HER2 at present, this makes to be difficult to make a distinction with the patient who only expresses HER2 expressing the patient of HER2 and CTFs.Compare with obtainable anti-HER2 antibody, antibody of the present invention is preferentially in conjunction with CTF-611, thereby can identify the patient who expresses this CTF.
The invention still further relates to the Method for cancer diagnostics of clinical samples, it comprises the existence that detects the HER2 acceptor clipped form being comprised of the aminoacid sequence shown in SEQ ID NO:1 in described clinical samples.
The present invention also provides described antibody or its fragment diagnosis and prognosis in separated sample to detect the application in the sample of expressing HER2 acceptor.
The present invention further provides the reagent that detects the cancer of expressing HER2 acceptor and/or C-end fragment for the sample diagnosis and prognosis from separated, it comprises at least one above-mentioned antibody or its fragment.
The present invention is also provided for the test kit that the cancer of HER2 acceptor is expressed in diagnosis and prognosis detection from separated sample, and it comprises the device existing for detection of the HER2 acceptor clipped form being comprised of SEQ ID NO:1 in described sample.
The present invention also provides above-mentioned antibody or the application of its fragment in treatment or pharmacy.Especially, this medicine is used for the treatment of or the cancer of the expression HER2 acceptor clipped form that prevents to be at least comprised of SEQ ID NO:1.
According to a further aspect in the invention, described antibody or its fragment, for the preparation for the treatment of or prevention Mammals, comprise the medicine of human breast carcinoma.
Another object of the present invention is to provide the pharmaceutical composition that is used for the treatment of the cancer of at least expressing the HER2 acceptor clipped form being comprised of SEQ ID NO:1, and it contains above-mentioned antibody or its fragment and at least one pharmaceutical excipient and/or carrier.
Accompanying drawing explanation
Fig. 1: the protein sequence of clipped form CTF-611, contrast figure with complete HER2 receptor sequence and the sequence of clipped form p95 (648-CTF/p95), it is the product of alpha-secretase zymoprotein hydrolysis.Cross-film functional domain represents spiral-line.All the other molecules show with grey frame.For generation of monoclonal antibody or how anti-peptide sequence, with underscore, represent.The position of intramolecular disulfide bond indicates with the connection between halfcystine.
Fig. 2: the electrophoresis of mammary cancer sample and western blotting (transferring on film).(S) represent soluble part, (M) be membrane portions.For detecting complete HER2 and CTF-611 form, use the antibody that directly acts on this albuminous cell matter functional domain.DHL: serum lactic dehydrogenase (with comparing).
Fig. 3: the measurement result that the transgenic mice quantity of expression CTF-611 clipped form (Fig. 3 A) tumour is compared with the transgenic mice of expressing complete HER2 acceptor; And the gross tumor volume result detecting.(Fig. 3 B).In Y-axis, N and V represent respectively tumour quantity and gross tumor volume.In X-axis, T represents the time (week).
Fig. 4: electrophoresis and western blotting (transferring on the film) result of the polyclonal antibody that Fig. 4 A produces for the antibody (CB11) of identification HER2 recipient cell kytoplasm functional domain (functional domains in all complete and clipped forms) or the polypeptide of anti-SEQ ID NO:3 (α-611-A and α-611-B).Express the NCF7 lysis extract vestige of HER2 acceptor complete form, CTF-611J clipped form and p95 clipped form.Fig. 4 B is the immunoprecipitation test-results that antibody α-611-A, α-611-B and CB11 act on the sample that contains complete HER2 acceptor and clipped form CTF-611 and p95.
Fig. 5: (A) HER2 is expressed in cracking, the MCF7 cell of 611-CTF or 648-CTF also carries out western blot analysis with acting on the antibody CB11 of HER2 tenuigenin functional domain or acting on two independent monoclonal anti-611-CTF antibody that polypeptide MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC (seeing Fig. 1) produces.(B) express HER2, the MCF7 cell lysate of 611-CTF or 648-CTF mixed with 1: 1: 1, and carried out immunoprecipitation by the monoclonal antibody of pointing out.Throw out and CB11 antibody after washing are carried out to Western hybridization analysis.As negative control, do not add antibody (-) and carry out immunoprecipitation.(C) in Laser Scanning Confocal Microscope, analyze and express HER2, the MCF7 cell of 611-CTF or 648-CTF reacts with the indirect immunofluorescence between the antibody showing.
Fig. 6 (A) and (B): a kind of monoclonal anti body characteristics that adopts Flow cytometry to arrive.
Fig. 7: the monoclonal anti body characteristics that adopts immunohistochemistry to detect.
Fig. 8: the feature of epitope.Fig. 8 (A): the primary sequence of pictorialization 611-CTF membrane-proximal region and the different sequence forming are for epitope mapping.Fig. 8 (B) western blotting.
Fig. 9: mammary cancer sample analysis.Fig. 9 (A) analytical results form.Fig. 9 (B) immunocytochemical stain.
Detailed Description Of The Invention
CTF-611
As previously mentioned, contriver is surprised to find the difference detected result of HER2 clipped form of described SEQ ID NO:1 and the mammary tissue cancer of the general type of poor prognosis has good dependency.Therefore, Fig. 3 has shown the measurement result of the transgenic mice quantity of expression clipped form CTF-611 (SEQ ID NO:1) tumour.
In order to obtain the result of Fig. 3, cDNA construct is expressed in the epithelium of mouse, and described cDNA construct encoding human SEQ ID NO:1 (CTF-611) is M611-CTF in figure.This cDNA construct is included in the SEQID NO:1 under the control of mouse mammary tumour virus (MMTV) promotor.In contrast, use mouse cell FVB/N-Tg (MMTVneu) 202J that expresses HER2 complete form, it is also under the control of MMTV promotor.Known from Fig. 3 A, in mouse, expression, with the tumour quantity of CTF-611 form (SEQ ID NO:1) cDNA construct far above one in FVB/N-Tg (MMTVneu) 202J cell, is HER2/neu in figure.The tumour of transgenic animal that detects the expression CTF-611 in 17 week age, its representative takes out FVB/N-Tg (MMTVneu) 202J mouse first 3 months first.It is stronger that this fact shows to express the tumor invasiveness that the tumour of HER2 fragment expresses complete forms than those.It is much bigger that Fig. 3 B also shows to express those animal tumor volumes of expressing the complete acceptor of HER2/neu of animal tumor volume ratio of CTF clipped form of the HER2 with SEQ ID NO:1.Second fact also shows that such tumor invasiveness is stronger.Contriver has also determined that the tumour of the expression HER2/neu that the tumour of those expressed sequences SEQ ID NO:1 clipped form is observed than those has higher lung's shift index.The experiment providing in embodiment 9 below has further proved clinical correlation.
All these transgenic animal data have disclosed to be had the HER2 acceptor clipped form of SEQ ID NO:1 and the difference between the complete form of this receptor and detects and be very important.
Obviously, described SEQ ID NO:1 comprises all from the allelic mutant between individuality, and it comprises the variation in some amino acid quantity and type, for example general 2 or 3 amino acid, or an amino acid is by another amino acid substitution, it does not change the one-piece construction of acceptor.
These HER2 clipped forms can comprise neoantigen epi-position, and in other words, new antigenic determinant is not present in complete acceptor, show that they are different with complete acceptor (antibody, medicine etc.) from the interaction mode of molecule.
CTF-611 antibody
Contriver can prepare polyclonal antibody and the monoclonal antibody of CTF-611.The epitope that the sequence that these antibody recognition comprise in SEQ ID NO:2 limits, the preferably epitope of SEQ ID NO:3 or its sequence definition comprising.More preferably identify the antibody of the epitope of MPIWKFPDEEGAS (SEQ.ID NO:5) or its sequence definition comprising.
In the present invention, epitope is understood to the part of peptide family macromolecule (or antigen), and its sequence and/or sterie configuration can be by immune system recognition (antibody, T cell, B cells).
Preferably antibody of the present invention can separate the albumen of SEQ ID NO:1 and HER2 receptor area.And preferably antibody of the present invention can come the albumen of SEQ ID NO:1 and HER2 acceptor clipped form 648-CTF/p95 difference.Can pass through one or more in the immunohistochemistry of immunofluorescence, flow cytometry, culturing cell and the immunohistochemistry of clinical samples described difference is visual.Particularly preferably with flow cytometry and immunoprecipitation, quantitatively distinguish.
According to the present invention, in immunoprecipitation experiment, preferred antibodies 611-CTF of the present invention exceeds at least 300 times than them in conjunction with HER2, more preferably at least 1000 times, most preferably 1000-2000 doubly, is specified in (with 5 microgram 32H2 and 5 microgram 20F4) in embodiment 3.
As mentioned above, antibody can be polyclonal or monoclonal.
In the present invention, " polyclonal antibody " should be understood to mean the one group of antibody being produced by different B cells (bone-marrow-derived lymphocyte).Polyclonal antibody is the mixture by immunoglobulin secretion for a certain antigen (polymer), and owing to being that B cell by different produces, any in these immunoglobulin (Ig)s can both be identified different epitopes.Therefore, in being contained in the different immunoglobulin (Ig) colony of polyclonal antibody, there is an immunoglobulin like protein, the target epi-position of its identification specific antigen epi-position or specific antigen.
For example, polyclonal antibody can be by the polypeptide of SEQ ID NO.2, SEQ ID NO.3, SEQID NO:4 or SEQ ID NO:5 is prepared as keyhole-limpet hemocyanin (Keyhole-limpet hemocyanin) conjugation is connected with immunogen, and with this conjugate immunity White Rabbit.Monoclonal antibody can be used same or analogous immunogen to prepare.The preparation of hybridoma cell line adopts method known to those skilled in the art.Then, hybridoma cell line is incubated in applicable substratum, reclaims the monoclonal antibody producing.
In a preferred embodiment, they are by according to budapest treaty, the preserving number (accessnumber) that is preserved in " German microbial strains preservation center-DSMZ " on April 9th, 2008 is for the hybridoma cell line of DSM ACC2904 or by according to budapest treaty, and the hybridoma cell line that the preserving number that is preserved in " German microbial strains preservation center-DSMZ " on November 6th, 2008 is DSMACC2980 prepares.
The monoclonal antibody particularly preferably preparing with these hybridoma cell lines and the monoclonal antibody at least with same function.At least there is same function and refer to that those can be similarly or distinguish better the antibody of CTF-611 and HER2.
Applicable antibody also comprises humanization (humanized) antibody and human antibody (human antibodies).
Therefore, the present invention also provides the polypeptide of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO:4 or SEQ ID NO:5 that separation obtains.
According to the present invention, can use antibody fragment, but not complete antibody.The group that described antibody fragment selects free F (ab), F (ab ') and Fv to form.In the present invention, antibody fragment refers to a part for antibody, and it has enough length and applicable structure is present in the epitope in HER2 acceptor clipped form with combination, thereby implements sample detection.
Embodiment 2 and 3 has provided concrete antibody.Obviously, the present invention also comprises that other directly acts on polyclone or the monoclonal antibody of the CTF-611 clipped form epitope of SEQ ID NO.2 and SEQ ID NO.3 restriction, and therefore, according to instruction of the present invention, they can directly be drawn by those skilled in the art.Similarly, the present invention includes the fragment of described antibody, such as F (ab), F (ab '), Fv etc., or any hybridoma cell line that can produce the monoclonal antibody of the polypeptide that can directly act on SEQ ID NO.2 and SEQ IDNO.3.
Diagnostic method
According to diagnostic method of the present invention, the existence that detects HER2 acceptor clipped form can adopt respectively following method to carry out: for the complete form of described HER2 acceptor, the differential migration in moving phase-stationary phase system by the HER2 acceptor clipped form that is comprised of the aminoacid sequence shown in SEQ ID NO:1 detects; And be combined and detect by the specific antibody of the HER2 acceptor clipped form with containing SEQ ID NO:1.
In the present invention, by differential migration, detect and be interpreted as any analytical technology that the mobility based on different testing compounds is carried out separation in specific moving phase-stationary phase system, for example electrophoresis.This different mobility may by compound with different electric charges, different molecular weight or the different avidity of other compound is caused.
This difference detects can be by analytical technology known in the art, and such as electrophoresis, molecular exclusion chromatography, antibody is affine test, immunofluorescence, immunocytochemistry, immunohistochemistry, flow cytometry etc. implemented.Immunohistochemistry particularly preferably.Just one or more methods combinations are to strengthen reliability.
The HER2 acceptor clipped form being comprised of SEQ ID NO:1, the difference also referred to as CTF-611 detects in the present invention, with regard to whole or complete HER2 acceptor, can make two kinds of different molecular weights and not homotactic albumen in Fig. 1 visual.The albumen that clipped form shown in SEQ ID NO:1 is obtained by 611 initial translations of methionine(Met) selectivity from HER2 acceptor complete sequence forms.Therefore this form or CTF contain new ectodomain, transmembrane segment and kytoplasm functional domain.Transmembrane segment is identical with the complete form of HER2 acceptor with kytoplasm functional domain.According to another clipped form of Fig. 1, be p95 or CTF-648.This form is the product that alpha-secretase enzyme acts on complete HER2 acceptor, and its major part by ectodomain is separated.
According to another embodiment of the invention, the step that detects the HER2 acceptor clipped form existence being comprised of the aminoacid sequence shown in SEQ ID NO:1 in sample comprises uses at least one above-mentioned antibody to detect.In a specific embodiment, diagnostic method according to the present invention comprises the epitope that detects SEQ ID NO:3 restriction.
In one embodiment of the invention, antibody of the present invention or its fragment are used as the reagent that diagnosis and prognostic detects, or directly for example, with their peptide sequence marks (using radio isotope) separately or indirectly (for example by add combined with fluorescent reagent or in selective reaction substratum, by reaction, can produce the reagent of fluorescence), above-mentioned purpose is all in order to produce optical signal, if possible, carry out quantitatively, to detect existing of the HER2 acceptor clipped form that formed by SEQ ID NO:1 in sample.
In an identical manner, design contains at least by the diagnostic reagent of the antibody that directly or indirectly method connected with spacerarm is combined with solid support, such reagent can be captured the form of the SEQ ID NO:1 sequence of sample, uses subsequently other non-specific antibody (for example CB11) to detect its existence.
Diagnosis and prognostic detection reagent of the present invention also can be used for immunohistochemistry operation.For this purpose, mono-clonal or polyclonal antibody (one-level or secondary) need suitably mark to produce optical signal, under quantitative optimal cases, once they contact with test organization, they just can with target protein to be measured, namely the CTF fragment of the HER2 of SEQ ID NO:1 sequence is had an effect.
Analysis, diagnosis and prognostic for the ease of doctor detect, diagnostic reagent is designed to kit form, except antibody, it comprises the reaction reagent reagent of immunohistochemical staining (for example for), damping fluid and is suitable for detecting the detection solution that the clipped form of sample SEQ ID NO:1 exists, the contrast slide glass of the different expression levels of reflection CTF-611 and detailed explanation, assist doctor diagnosed evaluation.This test kit for example also comprises, for the method for sxemiquantitative Immunohistochemistry HER2 (Herceptest tM).
Therefore, the invention provides the test kit detecting for diagnosis and prognostic, except reaction reagent well known in the art and be suitable for antibody and damping fluid that epitope reacts, it at least comprises a kind of antibody or its fragment, and described antibody or fragment can be identified the epitope being defined by SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5.
Diagnostic method of the present invention and test kit can be predicted nodus lymphoideus transferring rate, poor prognosis and anti-Herceptest tM.
For the complete form of HER2 acceptor, another kind of types of kits of the present invention comprises the device being necessary that the differential migration of the HER2 acceptor clipped form by being comprised of the aminoacid sequence shown in SEQ ID NO:1 detects.
In expressing the cancer group of HER2 acceptor or its clipped form, mammary cancer is the most outstanding.Other cancer is also expressed these albumen, comprise lung cancer, carcinoma of the pancreas, large bowel cancer, cancer of the stomach, prostate cancer, incidence cancer, skin carcinoma, kidney, carcinoma of testis, thyroid carcinoma, bladder cancer, uterus carcinoma, carcinoma of vagina, carcinoma of endometrium, ovarian cancer, esophagus cancer, oral carcinoma, salivary-gland carcinoma, laryngocarcinoma, peritoneal cancer, rhinocarcinoma and laryngocarcinoma, carcinoma of fallopian tube, kidney parent cell cancer, lymphatic cancer, and swing sarcoma, synovial sarcoma, medulloblastoma, trophocyte's knurl, glioma, glioblastoma, cholangiocarcinoma, cholesteatoma, chondrosarcoma, ependymoma, schwannoma, neuroma, rhabdosarcoma.Therefore, diagnostic method of the present invention is particularly useful for diagnosing any in these cancers.Except vitro detection CTFS, also can be by antibody for in-vivo imaging.
Methods for the treatment of
Antibody of the present invention or its fragment also can be used for treatment, particularly cancer therapy, and particularly those express the cancer of HER2 acceptor or its clipped form.Preferred humanization and human antibody and fragment thereof.
Antibody of the present invention also can be used for the pharmaceutical composition that the HER2 acceptor clipped form cancer being comprised of the aminoacid sequence shown in SEQ ID NO:1 is at least expressed in treatment.Said composition contains pharmaceutical carrier or vehicle and at least one identification by the epitope of SEQ ID NO:2 or SEQ ID NO:3 sequence definition.This pharmaceutical composition also comprises mixtures of antibodies, except antibody of the present invention, contains the antibody that other acts on HER2 or its fragment, for example Herceptest tM.
Based on accompanying drawing of the present invention and exemplary illustration but be not limited to embodiment, below described diagnosis and prognostic and detected the method for expressing HER2 acceptor and/or its C end fragment (clipped form) cancer; New polypeptide and act on the specific antibody of described polypeptide; New cell strain; Detect with diagnostic reagent and test kit, all these are the object of the invention.
Although do not illustrate, the technology of using in the present invention and scientific terminology are well known.Method and material similar or that be equal to can be used for enforcement of the present invention.In specification sheets and claim, " comprise (comprise) " and the version of this word, for example, " comprise (comprising) " and can not get rid of other technical characterictic, additive, component or step.Based on specification sheets or implement the present invention, other object of the present invention, benefit and feature will be apparent to those skilled in the art, and the embodiment below exemplifying and accompanying drawing are not used for limiting the scope of the invention.
In addition, should understand the combination that the present invention includes all above-mentioned preferred and specific group.
Embodiment
Following examples have been described from the cancer sample of separated expression HER2 acceptor and/or its clipped form, detect the different methods of the clipped form existence of sequence SEQ ID NO:1.
testing sequence
Cell: by MCF7Tet-Off cell (BD bio-science) be placed in contain 10%FBS (Gibco), 4mM L-glutamine (PAA Laboratories), 0.2mg/ml G418 (Gibco) and 1 μ g/ml doxycycline (Sigma) DMEM/F-12 (1: 1) (Gibco), at 37 ℃, under the condition of 5%CO2, preserve.Use FuGENE6 (Roche), with various expression plasmid transfectional cells.With hygromycin B (Invitrogen) screening of 0.1mg/ml, contain the single stable clone based on integrated plasmid pUHD10-3h.By removing the pUHD10-3h of the cDNAs of doxycycline induction coding HER2 and CTFs, express.First use 0.5% trypsinase-EDTA (GIBCO) isolated cell, by centrifugal, wash 3 times, in culture dish, after inoculating cell 10h, change substratum.To act on the antibody of HER2 tenuigenin functional domain, pass through each cell strain of confocal immunofluorescence microscopy microscopic examination homology.Pick out the stable cell strain of two strains for test.
Western blotting: RIIPA damping fluid (the 20mM NaH changing 2pO 4/ NaOHpH7.4,150mM NaCl, 1%Triton X-100,5mM EDTA, 100mM PMSF, 25mM NaF, 16 μ g/ml Trypsin inhibitor,Trasylols, 10 μ g/ml leupeptins and 1.3mM Na 3vO 4) in the cell of expressing different HER2 hypotypes is carried out to cracking, with DC protein assay reagent (BIO-RAD), measure protein concentration.By sample and the sample loading buffer that contains 5% beta-mercaptoethanol (final concentration: 62mM Tris pH6.8,12% glycerine, 2.5%SDS) mixes, and cultivates 5min at 99 ℃, then carries out SDS-PAGE, isolates 15 μ g albumen.Software I mageJ 1.38 for distinctive signal in western blotting (NIH) carries out quantitatively.
Immunoprecipitation: cell lysate is cultivated to 1h from different antibody at 4 ℃.Then, with albumin A, purify immunocomplex.With lysis buffer washing immunoprecipitate 3 times, mix with sample loading buffer and carry out western blot analysis.
The cell for immunofluorescence microscopy observation that is inoculated in cover glass is washed with PBS, with the fixing 20min with 0.2%Triton X-100 permeabilized processing 10min of 4% paraformaldehyde.With containing 1%BSA, 0.1% saponin and 0.02%NaN 3pBS seal (blocking) and antibodies, with Vectashield (Vector laboratories) mounting of DAPI.
Flow cytometry: separated in expressing the MCF7 cell of HER2,611-CTF or 648-at 4 ℃ with PBS washing and containing the PBS of 5mM EDTA.Separated cell and the anti-32H2 monoclonal antibody of 10 μ g/ml are being cultivated to 30min containing in the PBS of 5%BSA, in 4 ℃ of washings and in 4 ℃ of anti-mouse IgG (Becton-Dickinson) by FITC-conjugation containing the 30min that dyes in the PBS of 5%BSA, on FACscan, carry out flow cytometry, use FACscan research software (Becton Dickinson Immunocytometry Sys., Mountain View, CA).
Immunohistochemistry:. separated in expressing the MCF7 cell of HER2,611-CTF or 648-at 4 ℃ with PBS washing and containing the PBS of 5mM EDTA.Then, centrifugal, fixed cell throw out in 10% neutral formalin, dewaters and is embedded in paraffin.By the section from cell precipitation thing or from 4 μ m, thick tissue section is placed on the coated slide glass of poly-lysine.By operating below and carrying out immunohistochemical analysis:
1. de-paraffin and rehydration section
1.1 cultivate slide glass 30min in 60 ℃.
1.2 take off paraffin 3 times to slide glass, each 5min.With clean dimethylbenzene mixed culture, then with the pure washing of ethanol analysis 2 times, each 3min.
1.3 are placed in distilled water gradually: alcohol 95 ℃ 3min, 70 ℃ of 3min of ethanol, 50 ℃ of 3min of ethanol, distilled water.
2. antigen reclaims
At low pH (6), carry out PT connection (Link), the high pH 10x.DM 812 of ENVISION FLEX TARGETRETRIEVAL SOLUTION.20min，95℃。
With Envision Flex lavation buffer solution washing x10DM 811,15min.
3. immunohistochemical staining
AUTOSTAINER adds Link DAKO
Test kit: ENVISION FLEX+MOUSE, high pH (Link):
Envision Flex peroxidase sealing SM801.
Envision Flex/HRP SM 802。
Envision Flex DAB+ chromogen DM 807.
Envision Flex substrate buffer solution SM 803.
Envision Flex lavation buffer solution 10x DM 811.
Envision Flex target reclaims the high pH 10x of solution DM 812.
Envision Flex+ mouse (linker) SM 84.
Add peroxidase 5min and wash with lavation buffer solution.
Protein block 5%15-20min.
With lavation buffer solution, wash.
■ 32H2 wash-out 1: 1000-1: 3000 (1mg/ml mother liquor) or 20F4 wash-out 1: 20-1: 50 (1mg/ml mother liquor) 2 hours.
■ washs with lavation buffer solution.
■ secondary (Flex HRP, Flex+ mouse/rabbit) 20min.
■ washs with lavation buffer solution.
■DAB 5min。
■ washs with lavation buffer solution.
■ phenodin.
■ distilled water wash.
3. dewater and fix with mounting medium
The ethanol that 3.1 use concentration increase progressively carries out gradient washing (50%, 70%, 95%, at every turn wash 2min).
3.2 are placed in distilled water (alcohol 95 %, 70%, 50%, distilled water washs 3min at every turn) successively.
3.3 use dimethylbenzene/eucalyptol washing (washing each 2min 3 times).
3.4 use DPX sealing.
Transgenic mice
TG 611 and TG 687 mouse pass through gene engineering method, by the sequence clone of coding 687-CTF and 611-CTF, to pMB carrier, (Dr.Marcos Malumbres is so kind as to give, CNIO, Madrid) the multiple clone site II in the downstream, mouse mammary tumour virus long terminal repeats that strengthens of Rous sarcoma virus.Basis system (Founder lines) obtains by microinjection linear plasmid DNA in the ovocyte to fertilization, the super row FVB mouse that described ovocyte provides from Animal Biotechnology and gene therapy center (Centre de Biotecnologia Animal i Ter à pia Genica, Universitat Aut ò noma de Barcelona).By the genotype of Southern hybridization analysis basis mouse (founder mice).After basis animal is identified, conventional flora is by pcr gene somatotype.Male and female FVB/N-Tg (MMTVneu) 202J mouse is from Jackson laboratory (Bar Harbor, ME).
Entirely organize mounting (Whole Mounts) and histology
Mammals body of gland is embedded on slide glass, in 4% paraformaldehyde, fixedly spends the night and be transferred in 70% ethanol.The fuchsin solution that slide glass water is rinsed to 5min and the be placed in 0.2% filtration 24h that dyes.Then the ethanol continuously dehydrating that body of gland successively decreases by concentration, then degreasing being stored in wintergreen oil.By fixing body of gland sealing (blocked), in paraffin, section is also used for histologic analysis with h and E dyeing.
embodiment 1:by differential migration, detect SEQ ID NO:1 fragment.
Fig. 2 has shown that gel electrophoresis and Western-type shift the result of (western blotting), by different mammary cancer sample (108,114,101,103,131,134 and 145) application of sample on swimming lane.Analysis is from soluble part (S) and the membrane portions (M) of each sample cell lysate, makes visual and definite its composition of the type of HER2 molecule.Substantially, estimate that complete acceptor and SEQ ID NO:1 form are all present in cytolemma, in order to detect complete HER2 and CTF-611 form, use antibody (CB11) to directly act on the tenuigenin functional domain of described albumen, it is two kinds of functional domains that albumen form is total.As analysis of control, detect exist (DHL) of serum lactic dehydrogenase.In most of sample, there is band that complete HER2 acceptor is corresponding and in cytolemma part, occur the band of the CTF-611 form responding of SEQ ID NO:1.
Therefore, Fig. 2 shows to pass through the type of protein electrophoresis analyzing and testing HER2 acceptor clipped form.In addition, the existence of the HER2 fragment of sequence SEQ ID NO:1 represents specific cancer types, and as for embodiment, in expressing the cancer of HER2, mammary cancer should be taken as case and evaluate and process.
embodiment 2:use the polyclonal antibody of the epitope limiting for new SEQ ID NO:2 or SEQ ID NO:3 to detect the existence of the HER2 fragment of SEQ ID NO:1.
In order to detect the existence that sequence is the HER2 clipped form of SEQ ID NO:1, adopt other device to substitute the differential migration device in electrophoresis, the synthetic polypeptide with SEQ ID NO:4 sequence is also with 4 rabbits of described polypeptide immune.This polypeptide is corresponding to 32 amino acid of the N-end of CTF-611 form or SEQID NO:1; wherein most of halfcystine is replaced by Serine; make polypeptide can with immunogen succinylation key hole Qi hemocyanin (Keyhole-limpethemocyanin, KLH) conjugated.
Therefore, identical with SEQ ID NO:3, polypeptide corresponding to transformation SEQ ID NO:4 is to implement immunological technique.It will be understood by those skilled in the art that the antibody that directly acts on the synthetic polypeptide of SEQ ID NO:4 also can identify the epitope being defined by SEQ ID NO:3 in HER2 acceptor clipped form (SEQ ID NO:1 or CTF-611).Similarly, it will be understood by those skilled in the art that, if synthetic immune peptide is comprised of SEQ ID NO:2, it comprises all amino acid that is positioned at the CTF-611 acceptor of extracellular region, for identical object, the antibody that directly acts on other polypeptide that obtains being equal to.
To expressing, the MCF7 lysis extract of HER2 acceptor complete form, CTF-611 clipped form and p95 clipped form carries out SDS electrophoresis and follow-up Western shifts.Two fragments that the clipped form practical manifestation of SEQ IDNO:1 is similar molecular weight.As the experiment of carrying out with Glycosylase-F can draw, a kind of endonuclease capable reduces the N-polysaccharide (not shown) on albumen, and CTF-611 fragment is posttranslational modification substrate.Particularly, also enter subsequently Secretory Pathway carries out N-glycosylation to the fragment of synthetic about 110kDa.
The serum of two immune rabbits can be identified complete HER2 acceptor and CTF-611.From Fig. 4 A, can find out, complete HER2 acceptor and SEQ ID NO:1 or band corresponding to CTF-611 be detected.Fig. 4 A shows, western blotting result obtains identifying the antibody (CB11) of HER2 recipient cell kytoplasm functional domain (for complete form and the total functional domain of clipped form); Or obtain being present in the polyclonal antibody (α-611-A and α-611-B) being produced by polypeptide described in SEQ ID NO:4 in rabbit anteserum.Due to antibody α-611-A and the α-611-B signal corresponding to complete HER2 and CTF-611, know them by inference and can identify and be present in two kinds of linear epitopes in acceptor form.As the negative control of SDS electrophoresis and western blotting, use the MCF7 cell lysate of expressing p95 acceptor form, it does not have the epitope that acts on designed antibody.
These results relatively, when carrying out immunoprecipitation with antibody α-611-A and α-611-B and antibody CB11, detect and directly act on the antibody that the antibody of SEQ ID NO:4 polypeptide likens to for acceptor clipped form (CTF-611) and preferably precipitate.Be easy to form in the mixture of precipitation containing proportional be the multi-form cell lysate of expression HER2 acceptor of 1: 1: 1.These results in Fig. 4 B, have clearly been embodied.In figure, demonstrate SDS electrophoresis and western blotting and shift the band obtaining, these bands are that application of sample antibody α-611-A, α-611-B and CB11 carry out the result that immunoprecipitation test obtains.Contrast band (input) is with the cell lysate mixture application of sample of 1: 1: 1 that is easy to form with different antibodies the three types of immunoprecipitation.With CB11, carry out western blotting.Approximately 106 cell cracking in 500 μ l lysis buffers of expressing total length HER2,611-CTF or 648-CTF (50mM Tris HCL pH7.4,137mM NaCl, 2mM EDTA, 10% glycerine, 1%NP40).Lysate is clarified by the centrifugal 30min of 14000g.Input:5 μ l cleavage mixture (total length HER2: CTF611: CTF648,1: 1: 1).IP:50 μ l cleavage mixture+5 μ l is from the anti-611CTF serum of rabbit or CB11.
The respective strap obtaining with antibody α-611-A and α-611-B immunoprecipitation, the molecular weight of albumen band obtaining than the glycosylation of CTF-611 and non-glycosylated form is obviously darker, thereby can clearly make a distinction.That is, described in directly act on SEQ ID NO:4 polypeptide antibody can not with the complete form generation immunoprecipitation of HER2 acceptor, this shows that they accurately identify the epitope of the complete HER2 acceptor not changing.Therefore, known antibody α-611-A and α-611-B have specificity to CTF-611.
This species specificity is tested (not shown) by indirect immunoprecipitation and is verified, wherein affinity purification polyclonal antibody α-611-A and α-611-B only can make to express the sample dyeing of CTF-611 truncated segment.This true benefit is to use them to detect the multi-form possibility that becomes that is expressed in the HER2 acceptor in separated tumor tissues sample.The affinity purification operation of polyclonal antibody is as follows:
(i) with HiTrap albumin A HP post (GE Healthcare) purifying total IgG.Rabbit anteserum antibody is fixed on binding buffer liquid (Na 2hPO 4) on the post of balance, with the binding buffer liquid of 10 times of column volumes, wash and use citric acid (pH2.7) wash-out of 10 times of column volumes.With in the Tris-HCL of pH8 and elutriant.
(ii) with the polypeptide that is fixed on HiTrap NHS post, carry out purifying.Same polypeptide for immune rabbit is fixed on to HiTrap NHS post.The IgGs of purifying in above-mentioned steps is loaded onto with binding buffer liquid (Na 2hPO 4) on the post crossed of balance, with the binding buffer liquid of 10 times of column volumes, wash and use citric acid (pH2.7) wash-out of 10 times of column volumes.With in the Tris-HCL of pH8 and elutriant.
(iii) finally by the antibody of purifying PBS 0.02%NaN 3dialysis.
Complete HER2 acceptor comprises, the region of containing 6 disulphide bridgeses near cytolemma, represents with the wire between halfcystine in Fig. 1.The clipped form of CTF-611 or SEQ ID NO:1 sequence is only containing 5 halfcystines, the homodimer (homodimers) with the disulphide bridges that confirms to form between them in order to firm this clipped form.On the whole as can be known from Fig. 4, different from antigen region in complete HER2 acceptor near the cytolemma region of SEQID NO:1 clipped form.Accordingly, according to aforementioned content is known, carry out difference detection.
In input (input), different HER2 hypotypes is quantitative, according to standardized HER2 amount immunoprecipitation CB11 and anti-611-B, is listed in the table below:
Input CB11 Anti-611-B
HER2
1 1 1
611-CTF 1.47 1.27 23.20
648-CTF 1.53 2.76 2.4
With ImageJ 1.38, carry out quantitatively.
embodiment 3:by the monoclonal antibody that directly acts on the new epitope being defined by SEQ ID NO:2 or SEQ ID NO:3, detect the existence of the HER2 fragment of sequence SEQ ID NO:1.
Phase homopolypeptide with SEQ ID NO:4 in embodiment 2, obtains monoclonal antibody.In these all monoclonal antibodies, prepared by the hybridoma cell line that 20F4 is DSM ACC2904 by accession number, prepared by the hybridoma cell line that monoclonal antibody 32H2 is DSM ACC2980 by accession number.What with described antibody, obtain the results are shown in Fig. 5.
With method similar in embodiment 2, with 1: 1: 1 mixture of expressing the MCF7 lysis extract (using the thaumatropy of Fig. 1) of HER2 acceptor complete form or CTF-611 clipped form or p95 clipped form before, carry out SDS electrophoresis and Western transfer, in Fig. 5 A, Western film shows the result of CB11 or monoclonal antibody 20F4 and 32H2, the latter's specific recognition is corresponding to the HER2 clipped form of the CTF-611 acceptor of SEQ ID NO:1, , and can not detect the cross reaction with described acceptor complete form, this shows the new epitope (neo-epitope) that its accurate identification comprises the one-level amino on CTF-611 acceptor form N-end.
With similar method, with with antibody CB11 (the tenuigenin functional domain of its identification receptor), compare, when carrying out immunoprecipitation test with monoclonal antibody 20F4 and 32H2, the polypeptide that monoclonal antibody directly acts on SEQ ID NO:3 detected, the clipped form of single-minded precipitation acceptor (CTF-611).These results clearly illustrate in Fig. 5 B.In figure, shown with antibody 20F4,32H2 and CB11 and carried out the result application of sample that immunoprecipitation test obtains, carried out the result that SDS electrophoresis and Western shift.
Corresponding to the immunoprecipitation band of antibody 20F4 and 32H2, only can distinguish the band of glycosylation and the non-glycosylated form of CTF-611 clipped form.That is, antibody 20F4 and the 32H2 complete form of immunoprecipitation HER2 acceptor not.Therefore, known antibody 20F4 and 32H2 have high degree of specificity to CTF-611, nonrecognition HER2 acceptor complete form, show it can be used for distinguishing with high selectivity detect the specific form that is expressed in the HER2 acceptor in separated tumor tissues sample.
The feature that directly acts on the monoclonal antibody of 32 amino acid lengths of 611-CTF sequence N-end shows the existence of epitope, and it hides (masked) in total length HER2, but exposes in 611-CTF.These epitopes are hidden in soluble molecule and intact cell.In CB11,32H2 and 20F4, different HER2 hypotypes is quantitative, according to standardized HER2 amount, by immunoprecipitation, is listed in the table below:
CB11 32H2 20F4
HER2
1 1 1
611-CTF 1.01 343.50 1302.19
648-CTF 0.79 nd nd
embodiment 4:by flow cytometry, act on the feature of the monoclonal antibody of 611-CTF N-end
(A), with the monoclonal antibody 32H2 that acts on polypeptide MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC (seeing Fig. 1) of different concns, by flow cytometry, express the MCF7 cell of HER2,611-CTF or 648-CTF.See Fig. 6 (A).
(B) result of two routine tests in (A) carried out quantitatively and show mean value.See Fig. 6 (B).
(C) with the antibody of indicating, by indirect immunofluorescence, react, in Laser Scanning Confocal Microscope analysis, express the MCF7 cell of HER2,611-CTF or 648-CTF.Use Alexa Fluor 488 sheep anti-mouse iggs of Invitrogen (A110011) as the secondary antibody of fluorophor coupling.Without 32H2 serum but exist under the condition of secondary antibody (" 2ary ") and carry out FACS to detect non-specific binding, as negative control.
Conclusion: the epitope of antibody 32H2 identification is exposed in the pneumonocyte of expressing 611-CTF, but is hidden in the pneumonocyte of expressing HER2.
embodiment 5:by immunohistochemical analysis, act on the feature of the monoclonal antibody of 611-CTF N-end
With acting on the CB11 of HER2 tenuigenin functional domain or anti-611-CTF antibody that two are independently produced by polypeptide MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC, to expressing the MCF7 cell of HER2,611-CTF or 648-CTF, carry out immunocytochemical stain (seeing Fig. 1).The results are shown in Fig. 7.
Conclusion: by immunohistochemical analysis, the epitope of antibody 20F4 and 32H2 identification is exposed in the cell of expressing 611-CTF.By contrast, adopt these epitopes of constructed judgement to be hidden in total length HER2 molecule.This result show immunohistochemistry be clinical in the detection technique of conventional selection.
embodiment 6:act on the epitope feature of the monoclonal antibody identification of 611-CTF N-end
(A) diagram shows the primary sequence of 611-CTF membrane-proximal region.In figure, express N-end and the C-end of this molecule.Cross-film district and kinase function territory are indicated with the box of dotted line and grey respectively.Diagram shows different disappearance structure sequences.See Fig. 8 (A).
(B) the cracking cDNA disappearance structure transient transfection MCF-7 cell that starts coding from diagram amino acid.Cell lysate carries out western blot analysis with acting on the CB11 of HER2 tenuigenin functional domain or monoclonal anti-611-CTF antibody that two are independently produced by polypeptide MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC.See Fig. 8 (B).
Conclusion: contained or sequence coverage MPIWKFPDEEC. at least by the epitope of antibody 32H2 and 20F4 identification.
embodiment 7:with the monoclonal antibody or the Herceptest that act on 611-CTF N-end tManalyst's mammary cancer sample
(A) use Herceptest tMcarry out fluorescence in situ hybridization or western blotting, the expression of HER2 in analyzing samples.In the sample of judging for western blotting shown in figure, express can detection level HER2C end fragment (p95) (see (Scaltriti, M., Rojo, F., Ocana, A., Anido, J., Guzman, M., Cortes, J., Di Cosimo, S., Matias-Guiu, X., Ramon yCajal, S., Arribas, J., and Baselga, J. (2007) J Natl Cancer Inst 99 (8), 628-638)).See Fig. 9 (A).
(B) use the monoclonal anti-611-CTF antibody being produced by polypeptide MPIWKFPDEEGASQPSPINSTHSSVDLDDKGC, 32H2 or with the Herceptest that can make HER2 tenuigenin functional domain dyeing tMmammary cancer sample identical in (A) is carried out to immunocytochemical stain.See Fig. 9 (B).
Conclusion: by expressing with antibody 32H2 dyeing human breast carcinoma sample of being judged by western blotting before immunohistochemical analysis can detection level CTFs.
embodiment 8:setting up animal model characterizes the effect of CTF expression in body
With instrument, detect mouse model to determine the dependency of potential carcinogenic impatient HER2 in tumor development.In order further to determine its potential carinogenicity, we are based upon transgenosis (TG) mouse of expressing 611-CTF under mouse mammary tumor virus long terminal repeat (it shows active in mammary gland) control.Although this cell model shows that in extracellular soluble, CTFs is non-activity, in order further to study the result of these fragment expressions, we have also set up the TG animal of expressing 687-CTF.In contrast, we use classical and clearly model tormulation wild-type of feature HER2 (being that mouse is neural, rat neu).
7 week age, the 611-CTF level that is expressed in TG 611 heterozygosis and is F3 and F2 was respectively with the on level terms of endogenous HER2 and be 1/3rd of the level of endogenous HER2, yet the level of F1 is lower than detection threshold.Isozygotying of setting up is that the horizontal extent of middle 687-CTF is, is respectively TG687 and is the twice of HER2 level in F2 and F1 to half.
7 week age, the mammary gland of TG animal was not observed macroscopic abnormal.Yet the product red colouring morphologic detection of full mounting shows that in the tree-shaped latex dusts of TG 611 mouse, hyperplasia is abnormal.Although not obvious, in whole three systems of TG 611 mouse, occur similar abnormal.By contrast, the body of gland of TG687 mouse is compared not obvious with those wild-type mices.
embodiment 9:the expression of 611-CTF causes the development of malignant galactophore knurl
Although hyperplasia is more obvious in TG HER2 mouse, three tumour quantity, tumor growth and tumour generation aspects that tie up to every animal of TG 611 animals, have more invasive tumor.
Mammary gland
Figure BPA00001290536000241
(by the outward appearance of the monitoring of palpation weekly breast tumor.)
Even after 1 year, in TG 687 animals, do not observe tumour or abnormal yet.
Tumor histology analyzes and shows that HER2 induces typical aggressive solid nodositas cancer (invasive solid nodular carcinomas) in TG 611 mouse equally.Difference between the histology of the tumour being caused by HER2 and 611-CTF is only, in TG 611 mouse, the quantity of mitotic division imaging is higher.
As previously mentioned, TG HER2 mouse causes lung's transfer.Tumour detected after 3～6 weeks, approximately 1/4th TG HER2 animal lung can detect tumor nodule:
Figure BPA00001290536000242
(palpation is put to death small white mouse after tumour being detected after 3～6 weeks, monitor situation about shifting by immunohistochemistry.)
The histologic analysis that lung shifts has confirmed the expression of HER2, and has confirmed that with CK18 dyeing cell is from elementary tumour.More than the twice of the size of animal expression TG HER2 of the expression 611-CTF metastases detecting.This shows that the tumour being caused by CTF is more prone to invade lung.
sequence table
SEQ ID NO：1
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys
1 5 10 15
Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys
20 25 30
Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Val Ser Ala Val
35 40 45
Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly Ile Leu
50 55 60
Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg Arg Leu
65 70 75 80
Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Ala Met
85 90 95
Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu Arg Lys
100 105 110
Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile
115 120 125
Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Val
130 135 140
Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu
145 150 155 160
Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg Leu Leu
165 170 175
Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu Met Pro
180 185 190
Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg Leu Gly
195 200 205
Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly Met Ser
210 215 220
Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn
225 230 235 240
Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu
245 250 255
Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly Gly
260 265 270
Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg
275 280 285
Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu
290 295 300
Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arg Glu
305 310 315 320
Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile
325 330 335
Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp
340 345 350
Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg
355 360 365
Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp Leu
370 375 380
Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu Leu Glu
385 390 395 400
Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu Val Pro
405 410 415
Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly Gly Met
420 425 430
Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Asp
435 440 445
Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg Ser Pro
450 455 460
Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly Asp Leu
465 470 475 480
Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His Asp Pro
485 490 495
Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu Pro Ser
500 505 510
Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu
515 520 525
Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu
530 535 540
Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Ala
545 550 555 560
Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala
565 570 575
Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly
580 585 590
Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp
595 600 605
Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro Pro
610 615 620
Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu Gly
625 630 635 640
Leu Asp Val Pro Val
645
SEQ ID NO：2
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys
1 5 10 15
Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys
20 25 30
Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr
35 40
SEQ ID NO：3
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys
1 5 10 15
Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys
20 25 30
SEQ ID NO：4
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Ser Gln Pro Ser
1 5 10 15
Pro Ile Asn Ser Thr His Ser Ser Val Asp Leu Asp Asp Lys Gly Cys
20 25 30
SEQ ID NO：5
Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Ser
1 5 10
sequence explanation
Clipped form or the C end fragment (CTF) of SEQ ID NO:1 people HER2 albumen
The epitope of SEQ ID NO:2 people HER2 albumen clipped form
The epitope of SEQ ID NO:3 people HER2 albumen clipped form
SEQ ID NO:4 is by the synthetic polypeptide of people HER2 albumen clipped form epitope
SEQ ID NO:5 is by the synthetic polypeptide of people HER2 albumen clipped form epitope
Figure IPA00001290535500011
Figure IPA00001290535500021
Figure IPA00001290535500031
Figure IPA00001290535500051

Claims (24)

1. antibody or its fragment of identifying HER2 acceptor clipped form epitope, the sequence that described epitope is comprised by SEQ ID NO:2 limits, and wherein said antibody or its fragment are suitable for the albumen shown in SEQ ID NO:1 and the difference of HER2 acceptor to come.
2. antibody according to claim 1 or its fragment, the sequence that wherein said epitope is comprised by SEQ ID NO:3 limits.
3. antibody according to claim 1 or its fragment, wherein said antibody or its fragment be suitable for will the albumen shown in SEQ ID NO:1 and the upper difference of HER2 acceptor clipped form 648-CTF/p95 come.
4. according to the antibody described in claim 1 or 3 or its fragment, wherein can pass through immunohistochemistry in immunofluorescence, flow cytometry, culturing cell and one or more in the immunohistochemistry in clinical samples described difference is visual.
5. according to the antibody described in claim 1 or 3 or its fragment, it is polyclonal antibody.
6. according to the antibody described in claim 1 or 3 or its fragment, it is monoclonal antibody or its fragment.
7. antibody according to claim 6 or its fragment, it is by being preserved in DSMZ(Germany's microbial strains preservation center) preserving number be that the hybridoma cell line of DSM ACC2904 or hybridoma cell line that preserving number is DSM ACC2980 prepare.
8. according to the antibody described in claim 1 or 3 or its fragment, the group that wherein said fragment selects free F (ab), F (ab ') and Fv to form.
9. hybridoma, it prepares the monoclonal antibody described in claim 6 or 7.
10. prepare in claim 1-8 the method for antibody described in any one, comprise with the peptide of sequence shown in SEQ ID NO:3 or SEQ ID NO:4 inhuman object is carried out to immunization, it is optionally combined with immunogen.
11. methods for the preparation of monoclonal antibody according to claim 10, wherein hybridoma claimed in claim 9 ties up in suitable substratum and grows, and monoclonal antibody can reclaim and obtain from this substratum.
12. isolated peptides that formed by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.
Application described in 13. claim 1-8 any one in the test kit of antibody or its fragment cancer of diagnosis and prognosis detection expression HER2 acceptor the sample for the preparation of from separated.
14. detect the reagent of expressing HER2 acceptor or its clipped form types of cancer for the sample diagnosis and prognosis from separated, and it at least contains antibody or its fragment described in a kind of claim 1-8 any one.
15. diagnostic reagent according to claim 14, wherein antibody or its fragment are arranged on solid support.
16. detect the test kit of expressing HER2 acceptor or its clipped form types of cancer for the sample diagnosis and prognosis from separated, it comprises the device existing for detection of the HER2 acceptor clipped form being comprised of SEQ ID NO:1 in sample, and wherein said proofing unit at least contains the reagent described in a kind of claims 14 or 15.
17. test kits that detect for diagnosis and prognosis according to claim 16, the HER2 acceptor clipped form of wherein said proofing unit by the aminoacid sequence as shown in SEQ ID NO:1 with respect to as described in the differential migration of complete form of HER2 acceptor detect.
Antibody or its fragment in the claim 1-8 of 18. tumours for detection of expression HER2 acceptor clipped form described in any one.
19. antibody according to claim 18 or its fragment, it is for detection of the cancer of at least expressing the HER2 acceptor clipped form being comprised of the aminoacid sequence shown in SEQ ID NO:1.
20. according to the antibody described in claim 18 or 19 or its fragment, and it is for detection of mammiferous mammary cancer.
21. antibody according to claim 20 or its fragment, wherein said Mammals is behaved.
22. antibody according to claim 18 or its fragment, it is for detection of the cancer that is selected from lower group: lung cancer, carcinoma of the pancreas, large bowel cancer, cancer of the stomach, prostate cancer, incidence cancer, skin carcinoma, kidney, carcinoma of testis, thyroid carcinoma, bladder cancer, uterus carcinoma, carcinoma of vagina, carcinoma of endometrium, ovarian cancer, esophagus cancer, oral carcinoma, salivary-gland carcinoma, laryngocarcinoma, peritoneal cancer, rhinocarcinoma and laryngocarcinoma, carcinoma of fallopian tube, kidney parent cell cancer, lymphatic cancer, and swing sarcoma, synovial sarcoma, medulloblastoma, trophocyte's knurl, glioma, glioblastoma, cholangiocarcinoma, cholesteatoma, chondrosarcoma, ependymoma, schwannoma, neuroma, rhabdosarcoma.
23. antibody according to claim 19 or its fragment, it is for detection of the cancer that is selected from lower group: lung cancer, carcinoma of the pancreas, large bowel cancer, cancer of the stomach, prostate cancer, incidence cancer, skin carcinoma, kidney, carcinoma of testis, thyroid carcinoma, bladder cancer, uterus carcinoma, carcinoma of vagina, carcinoma of endometrium, ovarian cancer, esophagus cancer, oral carcinoma, salivary-gland carcinoma, laryngocarcinoma, peritoneal cancer, rhinocarcinoma and laryngocarcinoma, carcinoma of fallopian tube, kidney parent cell cancer, lymphatic cancer, and swing sarcoma, synovial sarcoma, medulloblastoma, trophocyte's knurl, glioma, glioblastoma, cholangiocarcinoma, cholesteatoma, chondrosarcoma, ependymoma, schwannoma, neuroma, rhabdosarcoma.
Antibody in 24. claim 1-8 described in any one or its fragment are being produced for detection of the application in the test kit of cancer.
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Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7638598B2 (en) * 2001-04-06 2009-12-29 The Trustees Of The University Of Pennsylvania ErbB interface peptidomimetics and methods of use thereof
CA2711843C (en) 2007-12-20 2018-11-13 Laboratory Corporation Of America Holdings Her-2 diagnostic methods
ES2342646B1 (en) * 2008-06-02 2011-04-26 Institut De Recerca Hospital Universitari Vall Hebron METHOD OF DIAGNOSIS OF CANCERES THAT EXPRESS THE RECEIVER HER-2 OR ITS TRUNCATED VARIANTS.
ES2637411T3 (en) 2008-12-01 2017-10-13 Laboratory Corporation Of America Holdings Methods and assays for measuring p95 Y / O p95 in a sample and antibodies specific for p95
BRPI1007321A2 (en) 2009-01-15 2018-07-10 Laboratory Corp America Holdings methods for measuring and / or quantifying the presence and / or amount of her-3 or her-3 in a complex in a patient sample, and to determine if an individual with cancer is likely to respond to treatment with a targeted therapy. and antibody.
NZ596468A (en) * 2009-05-14 2013-11-29 Nestec Sa Biomarkers for determining sensitivity of breast cancer cells to her2-targeted therapy
DK2330131T3 (en) * 2009-12-07 2015-01-05 Fundació Privada Inst D Investigació Oncol Gica De Vall Hebron Antibodies against HER2-truncated variant CTF-611
CN103102414B (en) * 2013-02-04 2014-08-27 无锡傲锐东源生物科技有限公司 HER2-resistant protein monoclonal antibody and application thereof
JP2016526016A (en) 2013-05-01 2016-09-01 ファイヴ プライム セラピューティクス インク How to treat cancer
JP6935195B2 (en) 2014-03-11 2021-09-15 モレキュラー テンプレーツ, インク.Molecular Templates, Inc. Protein containing binding region, effector region of Shiga toxin A subunit, and carboxy-terminal endoplasmic reticulum localization signal motif
US20160376328A1 (en) 2014-03-11 2016-12-29 Molecular Templates, Inc. Proteins comprising amino-terminal proximal shiga toxin a subunit effector regions and cell-targeting immunoglobulin-type binding regions
LT3303373T (en) 2015-05-30 2020-07-10 Molecular Templates, Inc. De-immunized, shiga toxin a subunit scaffolds and cell-targeting molecules comprising the same
KR20180030085A (en) 2015-07-26 2018-03-21 몰레큘러 템플레이츠, 인코퍼레이션. Cell-targeting molecule comprising a cytotoxin A subunit agonist and a CD8 + T-cell epitope
SG11201810032PA (en) * 2016-05-12 2018-12-28 Agency Science Tech & Res Anti-erbb-2 antibodies and uses thereof
ES2897217T3 (en) 2016-09-30 2022-02-28 Hoffmann La Roche Bispecific antibodies against p95HER2
CN110536695B (en) 2017-01-25 2024-05-10 分子模板公司 Cell targeting molecules comprising deimmunized shiga toxin a subunit effectors and cd8+ T cell epitopes
IL268443B1 (en) 2018-04-17 2024-03-01 Molecular Templates Inc Her2-targeting molecules comprising de-immunized, shiga toxin a subunit scaffolds
CN110172448B (en) * 2019-05-30 2020-07-28 中南大学湘雅二医院 Synovial sarcoma cell line hSS-005R and progeny cell line thereof
BR112022001148A2 (en) 2019-07-23 2022-03-15 Inst Nat Sante Rech Med Modified immune cells, pharmaceutical composition, kit, use of a modified immune cell and product invention
CN110865184B (en) * 2019-12-04 2023-04-07 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） Application of SRSP protein and SRSP epitope peptide and product for diagnosing and treating tumors
EP3915576A1 (en) 2020-05-28 2021-12-01 Fundació Privada Institut d'Investigació Oncològica de Vall-Hebron Chimeric antigen receptors specific for p95her2 and uses thereof
WO2023126458A1 (en) 2021-12-28 2023-07-06 Mnemo Therapeutics Immune cells with inactivated suv39h1 and modified tcr
EP4253418A1 (en) 2022-03-29 2023-10-04 Fundació Privada Institut d'Investigació Oncològica de Vall-Hebron Immune cells expressing chimeric antigen receptors and bispecific antibodies and uses thereof
EP4279085A1 (en) 2022-05-20 2023-11-22 Mnemo Therapeutics Compositions and methods for treating a refractory or relapsed cancer or a chronic infectious disease
WO2024062138A1 (en) 2022-09-23 2024-03-28 Mnemo Therapeutics Immune cells comprising a modified suv39h1 gene

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3040121B2 (en) 1988-01-12 2000-05-08 ジェネンテク，インコーポレイテッド Methods of treating tumor cells by inhibiting growth factor receptor function
WO1996006863A1 (en) 1994-08-30 1996-03-07 University Of Dundee Agents for inducing apoptosis and applications of said agents in therapy
WO1998006863A1 (en) * 1996-08-14 1998-02-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services A vector for polynucleotide vaccines
US7371376B1 (en) * 1996-10-18 2008-05-13 Genentech, Inc. Anti-ErbB2 antibodies
ZA9811162B (en) * 1997-12-12 2000-06-07 Genentech Inc Treatment with anti-ERBB2 antibodies.
EP1187632B1 (en) * 1999-05-14 2008-12-03 Genentech, Inc. TREATMENT WITH ANTI-ErbB2 ANTIBODIES
PL202369B1 (en) 1999-08-27 2009-06-30 Genentech Inc DOSAGES FOR TREATMENT WITH ANTI−ErbB2 ANTIBODIES
WO2005011607A2 (en) * 2003-08-01 2005-02-10 Smithkline Beecham Corporation Treatment of cancers expressing p95 erbb2
JP2006316040A (en) * 2005-05-13 2006-11-24 Genentech Inc Herceptin(r) adjuvant treatment
ES2342646B1 (en) * 2008-06-02 2011-04-26 Institut De Recerca Hospital Universitari Vall Hebron METHOD OF DIAGNOSIS OF CANCERES THAT EXPRESS THE RECEIVER HER-2 OR ITS TRUNCATED VARIANTS.
ES2637411T3 (en) * 2008-12-01 2017-10-13 Laboratory Corporation Of America Holdings Methods and assays for measuring p95 Y / O p95 in a sample and antibodies specific for p95

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Judit Anido等.Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation.《The EMBO Journal》.2006, *
Rosana Sáez等.p95HER-2 Predicts Worse Outcome in Patients with HER-2-Positive Breast Cancer.《clinical cancer research》.2006, *

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